Listeria monocytogenes is a facultative intracellular bacterial pathogen that causes serious illness in pregnant women, neonates, elderly, and immunocompromised individuals. Listeriosis is among the leading causes of death from contaminated food products in US. In the last decade, L.monocytogenes has served as an excellent model system for exploring the interactions that take place between an intracellular parasite and its host. The overall goal of this proposal is to define the mechanisms by which L.monocytogenes is capable of spreading from cell to cell without exposure to the extracellular environment. In previous studies, a broad-range bacterial phospholipase C (PC-PLC) was shown to be necessary for efficient bacterial cell-to-cell spread. PC-PLC is secreted as an inactive precursor (proPC-PLC), and proteolytic cleavage at its N-terminus generates the active form of the enzyme. Recently, we obtained genetic and biochemical evidence that the intracellular activation of pro PC-PLC is mediated by a bacterial metalloprotease (Mpl), which is also active in broth culture, and a cysteine protease, whose activity can only be detected during intracellular infection. The activity of PC-PLC generated by either protease is essentially the same, although there is a small shift in substrate preference. Furthermore, proPC-PLC activation by either pathway is dependent on bacterial localization to a vacuole, and on vacuolar acidification. These observations support a model of bacterial escape from double membrane vacuoles formed during cell-to-cell spread that is dependent on host and bacterial determinants. In this proposal, a multidisciplinary approach will be used to test this model of bacterial cell-to-cell spread. Intravacuolar activation of proPC-PLC will serve as a probe to define the host and bacterial requirements for efficient and rapid lysis of double membrane vacuoles. More specifically, this proposal will define (I) the vacuolar compartment in which proPC-PLC activation occurs, (II) the influence of other bacterial virulence determinants on vacuolar maturation and proPC-PLC activation, (III) the origin and identity of the intracellular-specific proPC-PLC activating cysteine protease, and (IV) the relative importance of the two activating proteases. The long term objective of this research is to define the mechanism by which L.monocytogenes spreads from cell to cell. This may provide a novel target for development of drugs to treat or prevent intracellular microbial infections in humans.